The human mitochondrial genome is an approximately 16 kilobase circular, double stranded DNA that encodes 13 polypeptides of the mitochondrial respiratory chain, 22 transfer RNAs, and two ribosomal RNAs required for protein synthesis. Multiple copies of mtDNA exist in a single cell.
The mitochondrial genome is particularly susceptible to mutations. It is believed that the elevated mtDNA mutation rate is a result of the high level of reactive oxygen species generated in the organelle, coupled with a low level of DNA repair. Mutations in the mtDNA sequence that affect all copies of mtDNA in an individual are known as homoplasmic. Mutations that affect only some copies of mtDNA are known as heteroplasmic, thus cells can contain a mixture of both mutant and wildtype mtDNA species. Since multiple copies of mtDNA exist in a single cell, a mtDNA mutation may only become physiologically relevant once the particular mtDNA mutation accumulates in the cell and exceeds a certain threshold.
Somatic cell mtDNA mutations have been intensely studied because of their proposed involvement in the development of diseases. For example, large heteroplasmic deletions of mtDNA have been identified in muscle biopsies from patients suffering from a relatively common mitochondrial myopathy. Missense and silent mutations in the genes encoding cytochrome c oxidase subunits have been shown to segregate with Alzheimer's disease and it has been demonstrated that a high proportion of human tumors contain one or more mtDNA mutations. It is known that the Pearson marrow-pancreas syndrome involves hematopoietic, as well as pancreatic, abnormalities arising from a defect of oxidative phosphorylation which is associated with deletions in the mtDNA. Finally, it is believed that the number of somatic mtDNA mutations in humans increases with the age of the individual and thus mtDNA mutations may be involved in the aging process.
Currently, there is no adequate method that allows the measurement of the mutational frequency in the mitochondrial or genomic (nuclear) DNA of intact animals, including humans. Thus, there remains a need in the art to develop methods for measuring and monitoring the mutational frequency of DNA in cells.